PROJECT SUMMARY This proposal unites complementary analytical expertise of Mayo Clinic and University of Wisconsin in pursuit of the goal of charting a molecular map of physical activity (PA). The overall goal of the Mayo Clinic Center for Metabolomics and Proteomics (MCCMAPS) is to apply state-of-the-art metabolomics and proteomics approaches to the MoTrPAC samples. We propose a metabolomics workflow that begins with untargeted profiling to identify candidate metabolites. From there, annotated metabolites will be validated, and unknown metabolites will be identified through a combination of novel compound synthesis, MS/MS data and NMR spectroscopy structural elucidation. The important metabolites that emerge from untargeted profiling will guide subsequent targeted quantitative metabolite measurements in plasma and tissue samples. We propose a proteomics workflow that includes global quantitative proteomics in muscle, adipose, and exosomes and assessment of posttranslational modifications using phosphoproteomics, acetylproteomics, and indices of oxidative modifications. Global, discovery proteomics will guide targeted, quantitative proteomics of salient proteins in plasma (e.g., irisin, myostatin). Our peptide synthesis capabilities allows for custom synthesis of any stable isotope labeled peptides for standards for quantitation. In addition, we will measure stable isotope enrichment of labeled amino acids in plasma, muscle compartments, and individual skeletal muscle proteins thus offering an insight on how PA influences amino acid kinetics, protein metabolism, and translation of gene transcripts to individual proteins. Isotopic enrichment of lipids in plasma and tissues will enable measurements of fatty acid kinetics in response to PA. Advanced statistical methods will fuse proteomics and metabolomics data to generate molecular concept maps of PA. Here we propose to leverage a productive ongoing collaboration between metabolomics and proteomics experts at Mayo and University of Wisconsin as a single integrated analytical resource dedicated to uncovering molecular signatures of PA. Our goal is to not only contribute our analytical expertise, but also contribute our knowledge of muscle and adipose tissue biology to the Molecular Transducers of Physical Activity Consortium (MoTrPAC).